Intractable seizure disorders in humans are often associated with cortical dysplasia, microgyria, and heterotopias resulting from neuronal migration disorders. We have used the rat freeze lesion model to examine neural mechanisms underlying hyperexcitability in focal cortical dysplasia. N-methyl-D-aspartate (NMDA) receptors are involved in generation of epileptiform discharges in this model and in human cortical dysplasia. The location of the NMDA receptors has not been determined and functional studies in human tissue are limited. Proposed studies will use electrophysiological and two-photon microscopy techniques to test specific hypotheses about the role of NMDA receptors in the freeze lesion model of cortical dysplasia. The applicability of these finding to human epilepsy will be directly tested in tissue slices from patients with focal cortical dysplasia using imaging methods. It is hypothesized that NMDA receptors are located on presynaptic nerve terminals of GABAergic interneurons in the early postnatal period and these receptors are normally developmentally downregulated but persist in cortical dysplasia. Experiments will investigate (1) if presynaptic NMDA receptors are present on inhibitory nerve terminals in neocortex. Whole-cell voltage-clamp recordings of pharmacologically isolated IPSCs will be obtained under conditions where postsynaptic NMDA receptors are blocked. It is hypothesized that NMDA receptor antagonists will decrease the frequency of miniature IPSC in young (PN 12-16) but not older (PN 26-30) sham operated animals. We will also use styryl dye (FM1-43) staining and multiphoton excitation microscopy to visualize vesicular release from inhibitory GABAergic terminals and study NMDA receptor modulation of GABA release. It will be determined if NMDA receptor activaton selectively modulates distinct vesicular pools, the readily releasable pool and the reluctant pool. We will also examine (2) if functional presynaptic NMDA receptors are present in human cortical dysplasia. Previous studies of human cortical dysplasia tissue has shown an increase in NMDA receptor expression, particularly NR2B receptors. It is hypothesized that NMDA receptors are located presynaptically on inhibitory nerve terminals and modulate vesicular release of GABA. These studies will provide important new information about presynaptic NMDA receptors and regulation of GABA release in cortical dysplasia. New insights into the role of presynaptic NMDA receptors in development and regulation of epileptiform activity will be forthcoming. These studies will also increase our understanding of basic mechanisms of transmitter release and its'developmental regulation in human and animal models of focal cortical dysplasia. PUBLIC HEALTH RELEVANCE: Cortical dysplasia is associated with intractable seizure disorders in humans. Up to 43% of patients receiving surgical treatment for intractable seizures have some sort of cortical malformation. Anticonvulsant drug therapy is often ineffective in these patients. Studies in vitro of brain slices prepared from human dysplastic neocortex have demonstrated that this tissue displays intrinsic hyperexcitability. The mechanisms responsible for the inherent epileptogenicity of dysplastic cortex have been incompletely defined. It is proposed to directly examine a previously unexplored mechanism, presynaptic NMDA receptors, using an animal model of cortical dysplasia and tissue samples from human cortical dysplasia. New insights into mechanisms regulating presynaptic release could lead to novel strategies for therapy.